MicroRNA-218-5p accelerates malignant behaviors of breast cancer through LRIG1

Highlights • MiRNA-218-5p up-regulates ErbB2 and EGFR expression by suppressing LRIG1 expression.• MiRNA-218-5p promotes the malignant behaviors of BC.• MiRNA-218-5p may exert a pro-tumor effect on BC and serve as a therapeutic target for BC treatment.


Introduction
MicroRNAs, a group of non-coding RNAs ranging 19-24 nucleotides, are capable of controlling the expression of targeted genes by inducing degradation or inhibiting the translation of mRNA.In recent years, plenty of evidence has indicated that microRNAs are implicated in a variety of cellular processes, such as proliferation, death, and metastasis [1−3].Moreover, microRNAs may also exert anti-tumor or protumor effects, depending on their expressing patterns and downstream targets involved [4,5].Some studies have revealed that miRNA-218-5p may act as an anticancer gene in various cancers, including renal cell carcinoma, hepatocellular carcinoma, gastric, colorectal and bladder cancers [6 −10].However, the exact underlying mechanism by which miRNA-218-5p orchestrates the pathogenesis of Breast Cancer (BC) remains unclear.
Leucine-Rich-Repeats and Immunoglobulin Like domains 1 (LRIG1) is widely expressed in many healthy tissues [11], and its expression is down-regulated in a number of carcinomas such as BC, renal carcinoma and cervical cancer [12−14].LRIG is an inhibitor of some oncogenic receptor tyrosine kinases, including ErbB family [14] as well as the Met [15] and Ret receptor [16] members.The physiological significance of LRIG1 has been underscored in LRIG1 knock-out mice which showed up-regulated expression of ErbB and Met receptor [17−19].
This study aimed to investigate the role of miRNA-218-5p in the malignant behaviors of BC.Our results showed that miRNA-218-5p expression was significantly elevated in BC tissues.miRNA-218-5p overexpression accelerated cell growth and metastasis of BC, disrupted cell cycle, and suppressed cell apoptosis by targeting LRIG1.These findings indicate that miRNA-218-5p may confer a pro-tumor effect in BC.

Collection of human BC tissues
Pathologically confirmed infiltrative breast ductal carcinoma tissues were collected from 30 patients from the Department of General Surgery of the Shanghai Tenth People's Hospital (Shanghai, China) and corresponding adjacent normal tissues were obtained as controls.Patients did not receive chemotherapy or radiotherapy before surgery.This study was approved by the Ethics Committee of Shanghai Tenth People's Hospital (20KT156) and informed consent was obtained before the study.Reporting guidelines are not applicable, because it is not a clinical study.

RNA extraction and RT-qPCR
A miRNA quick extraction kit (Tiangen, Beijing, China) was used to extract miRNA from BC tissues and adjacent normal tissues.Trizol reagent (Invitrogen, USA) was employed for isolation of total RNA RNA was reverse transcribed via a reverse transcription PCR kit (TaKaRa, Japan).A relative quantity of RNA was detected with a quantitative PCR (RT-qPCR) kit (TaKaRa, Japan).GAPDH was used as an endogenous reference.

Western blotting
Whole-cell proteins were extracted with protein lysis buffer (Sigma-Aldrich, USA) and quantified via a bicinchoninic acid assay (Pierce, USA).The proteins were separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membrane (EMD Millipore, USA).After blocking in non-fat milk, the membranes were incubated with antibodies against LRIG1-, ErbB2-or EGFR and then with horseradish peroxidase-conjugated secondary antibodies.Subsequently, an enhanced chemiluminescence detection kit (Millipore, USA) was employed for visualization.A Bio-RAD scanning system was applied to analyze the immunoreactive protein bands.

Migration assay
Cell migration was assessed via transwell assay (Corning, USA).In brief, cells were suspended in serum-free DMEM (180 μL) and then added into the upper chambers (6 × 10 4 per well).DMEM containing FBS (10%, 600 μL) was added to the lower chambers.16h later, 3% paraformaldehyde was added to fix cells on the polycarbonate membranes, and cells were stained with 0.1% crystal violet for 15 min.A cotton swab was used to collect cells in the upper chambers and migrate cells adherent to the base of the chambers.Images were captured under an inverted microscope (Thermo Fisher Scientific, USA) at 200 × and five fields were randomly selected for further analysis.

Flow cytometry
For cell cycle examination, after transfection with miRNA-218-5p inhibitor, miRNA-218-5p mimic or negative control, cells were collected and rinsed.Subsequently, ice-cold ethanol (3 mL) was added to cells, followed by incubation for over 30 min.After the addition of propidium iodide (PI; 0.05 g/L), cells were incubated at room temperature for 30 min, and then subjected to flow cytometry (Beckman coulter, USA).
Annexin V-FITC/PI cell apoptosis detection kit (BestBio, China) was employed for the detection of cell apoptosis.After transfection for 48h, cells were harvested, rinsed in PBS, and stained via Annexin V-FITC and PI.The percentages of apoptotic cells were determined by flow cytometer and analyzed using FlowJo software.

Statistical analysis
GraphPad Prism 5.0 software (GraphPad Software Inc., USA) was used for statistical analysis.Comparisons between two groups were performed with Student's t-test, whereas comparisons among groups were done with one-way analysis of variance.All experiments were carried out in triplicate, and data are expressed as mean ± Standard Deviation (SD).A value of p < 0.05 was considered statistically significant.

MiRNA-218-5p expression in human breast cancer
The expression of miRNA-218-5p was detected in 30 BC tissues and corresponding adjacent normal tissues via RT-qPCR.As shown in Figure 1A, as compared to the adjacent normal tissues, the miRNA-218-5p expression was markedly increased in BC tissues.In HCC1937, MDA-MB-231, MCF-7, and MDA-MB-468 cells, miRNA-218-5p expression was also detected.Results showed the miRNA-218-5p expression in BC cell lines was markedly higher than in MCF-10A cells, an immortal mammary epithelial cell line (Fig. 1B), which was consistent with findings

MiRNA-218-5p disrupts the cell-cycle progression of BC cells in different phases
The effect of miRNA-218-5p on the cell cycle progression was further investigated by flow cytometry.Results showed that miRNA-218-5p mimics transfection-arrested cell cycle in the G2/M-phase (Fig. 3 A-D).On the other hand, attenuation of miRNA-218-5p significantly elevated the proportion of cells in the S-phase and reduced that in the G2/Mphase (Fig. 3 A-D).These findings suggest miRNA-218-5p initiated S-phase arrest.

MiRNA-218-5p inhibits BC cell apoptosis
The apoptosis of BC cells after miRNA-218-5p up-regulation or down-regulation was further examined by flow cytometry.Results revealed that cells with miRNA-218-5p up-regulation showed a significantly lower proportion of late apoptotic cells as compared to the negative control (Fig. 4 A-D).On the contrary, miRNA-218-5p downregulation increased apoptotic cells, both in early and late apoptosis (Fig. 4 A-D).In summary, these indicate that miRNA-218-5p can inhibit BC cell apoptosis.

Discussion
Studies have reported that miRNAs can exert anti-tumor or protumor effects, and the expression of some miRNAs is altered during the development of tumors.[20−23] The dysfunction of miRNA in various cancers indicates that modulating miRNA expression may become a new therapeutic treatment for cancers.Up to now, miRNA-targeting therapy has been employed by using miRNA sponges, antisense oligonucleotides, or small-molecule inhibitors.Chemically synthesized miRNAs or oligonucleotides targeting miRNAs have been proven to efficiently inhibit cancer development.[24−27] At this time, several preclinical and clinical trials on miRNA-targeting therapy shedding light on cancer treatment are ongoing.[28−30] MiRNAs are vital for the tumor development of BC. [31−33] This study investigated the role of miRNA-218-5p in the pathogenesis of BC.Our results showed miRNA-218-5p expression in human BC tissues was markedly upregulated as compared to adjacent normal tissues.Similar findings have been reported in other cancers, indicating that increased miRNA-218-5p expression may be a common event in human cancers.[34−36] In the subsequent experiment, mimics of miRNA-218-5p were transfected into MCF-7 cells, and results showed that cell growth was markedly accelerated and cell migration was significantly promoted.All these findings indicate that miRNA-218-5p promotes the growth and migration of BC cells.In addition, miRNA-218-5p inhibited the cell cycle of BC cells.However, no marked difference was noted in apoptotic cells between miRNA-218-5p and negative control.
To reveal the underlying mechanism by which miRNA-218-5p exerts effects on cell growth, potential targets were searched in the miRNA Base, target scan, and miRNA and a database.LRIG1 was predicted to be a direct target of miRNA-218-5p, which was finally confirmed through luciferase reporter assay.In addition, LRIG1 mRNA and protein expression was dramatically lower in the miRNA-218-5p overexpression group than in the negative control group.These findings indicate that LRIG1 is a downstream target of miRNA-218-5p.
Studies have reported that LRIG1 can regulate ErbB family RTKs on cell surfaces.[37] In tamoxifen-treated luminal BCs, up-regulation of LRIG1 suppresses RTK family expression and signaling, including EGFR and ErbB2-4.[38] Our findings indicated that miRNA-218-5p up-regulated ErbB2 and EGFR expression by targeting LRIG1, suggesting that the LRIG1-mediated signaling pathway contributed to the effects of miRNA-218-5p on the malignant behaviors of BC.

Conclusions
Collectively, our study reveals that miRNA-218-5p may disrupt the cell cycle, induce cell growth and metastasis of BC cells via regulating LRIG1.These findings indicate that miRNA-218-5p may exert a protumor effect on BC.Furthermore, LRIG1 is a downstream target of miRNA-218-5p, and therefore decreasing miRNA-218-5p or upregulating LRIG1 may serve as new treatments for BC.